Objective: To investigate the relationship between runt-related transcription factor 2 (RUNX2) expression in canine nucleus pulposus (NP) cells and intervertebral disk aging in chondrodystrophoid dogs. Animals: 7 healthy Beagles (mean age, 35.6 months) and 11 Dachshunds with herniated disks (mean age, 61 months). Procedures: All dogs underwent MRI examination of the thoracic and lumbar vertebral column immediately before sample collection under general anesthesia. The disk center–to–CSF T2-weighted signal intensity ratio was determined for healthy Beagles. Samples of NP were obtained from nonherniated disks in healthy Beagles and from herniated disks during surgical treatment of hospitalized Dachshunds. Samples were evaluated for RUNX2 and matrix metalloproteinase 13 transcript expression via reverse transcriptase PCR assay; RUNX2 protein expression was evaluated via immunohistochemical analysis, and correlation between these variables and age of dogs was evaluated. A 3′ and 5′ rapid amplification of cDNA ends method was used to identify the RUNX2 coding region. Results: RUNX2 cDNA had > 97% conservation with the human cDNA sequence and approximately 95% conservation with the mouse cDNA sequence; RUNX2 and matrix metalloproteinase 13 mRNA expression and RUNX2 protein expression in NP cells were positively correlated with age. The disk center–to–CSF T2-weighted signal intensity ratio was negatively correlated with RUNX2 protein expression in the NP of healthy dogs. Conclusions and Clinical Relevance: Results indicated that RUNX2 mRNA and protein expression in the NP are enhanced in aging intervertebral disks in dogs.

Objective-To compare in vitro expansion of equine tendon- and bone marrow–derived cells with fibroblast growth factor-2 (FGF-2) supplementation and sequential matrix synthesis with pulverized tendon and insulin-like growth factor-I (IGF-I). Sample-Cells from 6 young adult horses. Procedures-Progenitor cells were expanded in monolayers with FGF-2, followed by culture with autogenous acellular pulverized tendon and IGF-I for 7 days. Initial cell isolation and subsequent monolayer proliferation were assessed. In pulverized tendon cultures, cell viability and expression of collagen types I and III and cartilage oligomeric matrix protein (COMP) mRNAs were assessed. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period. Results-Monolayer expansion with FGF-2 significantly increased the mean +/- SE number of tendon-derived cells (15.3 +/- 2.6 × 10(6)), compared with bone marrow-derived cells (5.8 +/- 1.8 × 10(6)). Overall, increases in collagen type III and COMP mRNAs were seen in tendon-derived cells, compared with results for bone marrow-derived cells. After IGF-I supplementation, increases in collagen type I and type III mRNA expression were seen in bone marrow–derived cells, compared with results for unsupplemented control cells. Insulin-like growth factor-I significantly increased collagen synthesis of bone marrow–derived cells. Monolayer expansion with FGF-2 followed by IGF-I supplementation significantly increased glycosaminoglycan synthesis in tendon-derived cells. Conclusions and Clinical Relevance-Tendon-derived cells had increased cell numbers and matrix synthesis after monolayer expansion with FGF-2, compared with results for bone marrow–derived cells. In vivo experiments with FGF-2-expanded tendon-derived cells are warranted to evaluate effects on tendon healing.

Objective-To compare in vitro expansion, explant colonization, and matrix synthesis of equine tendon- and bone marrow-derived cells in response to insulin-like growth factor-I (IGF-I) supplementation. Sample-Cells isolated from 7 young adult horses. Procedures-Tendon- and bone marrow-derived progenitor cells were isolated, evaluated for yield, and cultured on autogenous cell-free tendon matrix for 7 days. Samples were analyzed for cell viability and expression of collagen type I, collagen type III, and cartilage oligomeric matrix protein mRNAs. Collagen and glycosaminoglycan syntheses were quantified over a 24-hour period. Results-Tendon- and bone marrow–derived cells required 17 to 19 days of monolayer culture to reach 2 passages. Mean +/- SE number of monolayer cells isolated was higher for tendon-derived cells (7.9 +/- 0.9 × 10(6)) than for bone marrow–derived cells (1.2 +/- 0.1 × 10(6)). Cell numbers after culture for 7 days on acellular tendon matrix were 1.6- to 2.8-fold higher for tendon-derived cells than for bone marrow-derived cells and 0.8- to 1.7-fold higher for IGF-I supplementation than for untreated cells. New collagen and glycosaminoglycan syntheses were significantly greater in tendon-derived cell groups and in IGF-I-supplemented groups. The mRNA concentrations of collagen type I, collagen type III, and cartilage oligomeric matrix protein were not significantly different between tendon- and bone marrow-derived groups. Conclusions and Clinical Relevance-In vitro results of this study suggested that tendon-derived cells supplemented with IGF-I may offer a useful resource for cell-based strategies in tendon healing.

Objective: To isolate and characterize mesenchymal stem cells (MSCs) from canine muscle and periosteum and compare proliferative capacities of bone marrow-, adipose tissue-, muscle-, and periosteum-derived MSCs (BMSCs, AMSCs, MMSCs, and PMSCs, respectively). Sample: 7 canine cadavers. Procedures: MSCs were characterized on the basis of morphology, immunofluorescence of MSC-associated cell surface markers, and expression of pluripotency-associated transcription factors. Morphological and histochemical methods were used to evaluate differentiation of MSCs cultured in adipogenic, osteogenic, and chondrogenic media. Messenger ribonucleic acid expression of alkaline phosphatase, RUNX2, OSTERIX, and OSTEOPONTIN were evaluated as markers for osteogenic differentiation. Passage-1 MSCs were counted at 24, 48, 72, and 96 hours to determine tissue-specific MSC proliferative capacity. Mesenchymal stem cell yield per gram of tissue was calculated for confluent passage-1 MSCs. Results: Successful isolation of BMSCs, AMSCs, MMSCs, and PMSCs was determined on the basis of morphology; expression of CD44 and CD90; no expression of CD34 and CD45; mRNA expression of SOX2, OCT4, and NANOG; and adipogenic and osteogenic differentiation. Proliferative capacity was not significantly different among BMSCs, AMSCs, MMSCs, and PMSCs over a 4-day culture period. Periosteum provided a significantly higher MSC yield per gram of tissue once confluent in passage 1 (mean ± SD of 19,400,000 ± 12,800,000 of PMSCs/g of periosteum obtained in a mean ± SD of 13 ± 1.64 days). Conclusions and Clinical Relevance: Results indicated that canine muscle and periosteum may be sources of MSCs. Periosteum was a superior tissue source for MSC yield and may be useful in allogenic applications.

Objective: To evaluate use of a radiolabeled peptide nucleic acid–peptide conjugate (RaPP) targeting B-cell leukemia-lymphoma 2 (BCL2) mRNA for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma and to assess associations among RaPP uptake, time to tumor progression (TTP), and BCL2 mRNA expression. Animals: 11 dogs with B-cell lymphoma and 1 clinically normal dog. Procedures: Scintigraphic images were acquired 1 hour after IV injection of the RaPP. Regions of interest (ROIs) were drawn around lymph nodes, liver, and spleen; ROI intensity (relative to that of an equally sized region of muscle in the same image) was measured. Each ROI was also subjectively categorized as positive or negative for increased RaPP uptake. Expression of BCL2 mRNA was determined via quantitative reverse transcriptase PCR assay of a lymph node sample from dogs with lymphoma. Associations among imaging results, TTP, and BCL2 mRNA expression were evaluated. Results: Increased RaPP uptake was detected in affected tissues of dogs with lymphoma. Dogs with superficial cervical lymph node ROIs categorized as negative (n = 8) for increased RaPP uptake had a significantly longer TTP than did dogs for which this ROI was considered positive (2). Measured intensity of mandibular and superficial cervical lymph node ROIs was negatively associated with TTP. Associations among BCL2 mRNA and ROI intensity or TTP were not significant. Conclusions and Clinical Relevance: Increased RaPP uptake at mandibular or superficial cervical lymph node ROIs may be a negative prognostic indicator in dogs with lymphoma. A larger investigation is needed to determine clinical value of the RaPP for disease detection and prognostication.